Bicuspid aortic valve (BAV) is the most common type of congenital heart anomaly with an estimated incidence of 1-2% (Hoffman and Kaplan, 2002). While the presence of only two aortic valve leaflets instead of three may not be recognized early in life, progressive dysfunction of the valve is common. Age-related calcification of the aortic valve results in stenosis and insufficiency in one-third of affected individuals and also causes an elevated risk for infective endocarditis (Ward, 2000; Fedak et al, 2002). Calcium deposits in the valve are associated with increased incidence of coronary artery calcification and are more prominent in the setting of increased serum lipids, hypertension, and diabetes (Roberts, 1986; Stewart et al, 1997; Palta et al, 2000; Walsh et al, 2004). Obstruction of blood flow and regurgitation caused by an improperly functioning aortic valve results in an increased workload on the left ventricle and ultimately heart failure. Incomplete leaflet separation between two of the three valve leaflets is the underlying developmental defect, but pathogenesis of the long-term calcification has remained a mystery.
There is abundant evidence suggesting a significant inherited component to the etiology of BAV (Cripe et al, 2004). Children with hypoplastic left heart syndrome (HLHS) represent the most severe type of aortic valve obstruction, which results in failure of left ventricular growth during fetal life. 15-20% of relatives of children with HLHS have BAV, often undiagnosed, suggesting a common genetic etiology with phenotypic heterogeneity for these cardiac malformations (Loffredo et al, 2004). In addition, the congenital heart disease (CHD) recurrence risk for parents of children with HLHS is 10-15%, compared to 3-5% for most other CHDs (Nora and Nora, 1988; Whittemore et al, 1994). Based on such observations, an oligogenic cause for BAV and HLHS with other environmental or stochastic influences has been proposed, however the gene(s) that might contribute are unknown (Lewin et al, 2004).
The Notch1 gene encodes a 2556 amino acid protein that contains an extracellular domain with 36 tandem epidermal growth factor (EGF)-like repeats, three cysteine-rich Notch/LIN-12 repeats and an intracellular domain with six ankryin repeats and a transactivation domain. The Notch signaling pathway is highly conserved and has been well described (Artavanis-Tsakonas et al, 1999). The Notch receptor interacts with two ligands, Delta and Jagged (or Serrate in Drosophila). Ligand binding results in at least two independent cleavages of the Notch receptor, first by a metalloprotease (Wen et al, 1997; Sotillos et al, 1997), then by presenilin (Struhl and Adachi, 1998; Struhl and Greenwald 1999). Clipping of the protein results in release of the Notch intracellular domain (Notch IC) from the membrane similar to that first described for sterol response binding protein (SREBP) (Sakai et al, 1996). Notch IC translocates to the nucleus where it interacts with Suppressor of Hairless (Su(H); RBPJk) to activate downstream target genes, including members of the Hairy (enhancer of split) family of transcriptional repressors. This pathway is involved in cell fate determination and differentiation during organogenesis throughout the embryo and is regulated by glycosylation of the extracellular EGF-like repeats (Haines and Irvine, 2003). Disruption of Notch1 in mice results in embryonic lethality by E9.5 from vascular defects (Swiatek et al, 1994) but recent studies suggest that Notch1 is involved in cardiac epithelial-mesenchymal transformation in frogs and zebrafish (Loomes et al, 2002; Timmerman et al, 2004).
Here, we show that mutations in the signaling and transcriptional regulator Notch1 cause developmental aortic valve anomalies and premature valve calcification in autosomal dominant human pedigrees. We also found a Notch1 single nucleotide polymorphism (SNP) that resulted in an R1280H substitution present in two percent of the general population and demonstrated that this SNP conferred a nearly fifty percent risk for aortic valve calcification. Furthermore, we show that Notch1 normally represses the activity of Runx2, a central transcriptional regulator of the osteoblast cell fate, and that Notch1 repression of Runx2 in the valve was likely through Hairy-related transcriptional repressors belonging to the Hrt family of basic helix-loop-helix proteins. These results indicate that Notch1 mutations cause an early developmental defect in the aortic valve and a later de-repression of calcium deposition that result in progressive aortic valve disease. Furthermore, population genetics of Notch1 polymorphisms revealed individuals at increased risk for premature vascular calcification, identifying a large number that would benefit from early diagnosis and prevention.
Relevant Literature
Tezuka, K. et al., (J Bone Miner Res 17:231-9, 2002) report stimulation of osteoblastic cell differentiation by Notch.
Sciaudone et al. (Endocrinology. 144:5631-9, 2003) report that Notch 1 can impair osteoblastic cell differentiation.
Rajamannan, N. M. et al. (Circulation 107, 2181-4, 2003) report that human aortic valve calcification is associated with an osteoblast phenotype.
Zamurovic, N., et al. (J Biol Chem. 279:37704-15, 2004) report coordinated activation of notch, Wnt, and transforming growth factor-beta signaling pathways in bone morphogenic protein 2-induced osteogenesis, and that Notch target gene Hey1 inhibits mineralization and Runx2 transcriptional activity.
Zayzafoon, M., et al. (J Biol Chem. 279:3662-70, 2004) report Notch signaling and ERK activation are important for the osteomimetic properties of prostate cancer bone metastatic cell lines.